Quantitative assessment for cap efficiency of messenger RNA

ABSTRACT

The present invention provides, among other things, methods of quantitating mRNA capping efficiency, particularly for mRNA synthesized in vitro. In some embodiments, the methods comprise chromatographic methods of quantifying capping efficiency and methylation status of the caps.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is the U.S. National Stage entry from InternationalApplication No. PCT/US2014/027587, filed Mar. 14, 2014, which claimspriority to U.S. provisional patent application Ser. No. 61/784,337,filed Mar. 14, 2013, the disclosures of both of which are hereinincorporated by reference in their entireties.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has beensubmitted electronically in ASCII format and is hereby incorporated byreference in its entirety. Said ASCII copy, created on Jan. 18, 2018, isnamed MRT-1106-2US2_SL.txt and is 9,304 bytes in size.

BACKGROUND

Messenger RNA (“mRNA”) therapy is becoming an increasingly importantapproach for the treatment of a variety of diseases. Effective mRNAtherapy requires effective delivery of the mRNA to the patient andefficient production of the protein encoded by the mRNA within thepatient's body. To optimize mRNA delivery and protein production invivo, a proper cap are typically required at the 5′ end of theconstruct, which protects the mRNA from degradation and facilitatessuccessful protein translation. Therefore, accurate characterization ofthe capping efficiency is particularly important for determining thequality of mRNA for therapeutic applications.

SUMMARY OF THE INVENTION

The present invention provides improved methods for accurately andquantitatively determining the capping efficiency of mRNA, inparticular, mRNA synthesized in vitro. As discussed above, propercapping is important for successful protein production in vivo. However,prior to the present invention, most cap assays are qualitative, whichis not sufficient for assessing the quality of an mRNA based therapeuticproduct and related safety and efficacy for in vivo use. In fact, priorto the present invention, there is no method available that allowsquantification of capping efficiency without permanent alterations ofthe mRNAs in a sample.

As described in detail below, the present invention is, in part, basedon generation and quantification of capped and uncapped fragments bychromatography. Thus, the present invention provides a simple, reliableand efficient quantitative approach for assessing mRNA cappingefficiency. The present invention is particularly useful for qualitycontrol during mRNA manufacture and for characterization of mRNA as anactive pharmaceutical ingredient (API) in final therapeutic products.

In one aspect, the present invention provides methods of quantifyingmRNA capping efficiency, comprising steps of: (1) providing an mRNAsample comprising capped mRNA and uncapped mRNA; (2) contacting the mRNAsample with a DNA oligonucleotide complimentary to a sequence in the 5′untranslated region of the mRNA adjacent to the cap or uncappedpenultimate base of mRNA under conditions that permit the DNAoligonucleotide anneal to the sequence; (3) providing one or morenucleases that selectively degrade DNA/RNA hybrid and/or unannealedmRNA, resulting in capped and uncapped fragments; (4) separating thecapped and uncapped fragments by chromatography; and (5) determiningrelative amount of the capped and uncapped fragments, therebyquantifying mRNA capping efficiency.

In some embodiments, inventive methods of the present invention can beused to quantify a cap having a structure of formula I:

wherein,

B is a nucleobase;

R₁ is selected from a halogen, OH, and OCH₃;

R₂ is selected from H, OH, and OCH₃;

R₃ is CH₃, CH₂CH₃, CH₂CH₂CH₃ or void;

R₄ is NH₂;

R₅ is selected from OH, OCH₃ and a halogen;

n is 1, 2, or 3; and

M is a nucleotide of the mRNA.

In some embodiments, the nucleobase is guanine.

In some embodiments, inventive methods of the present invention can beused to quantify a m⁷G cap with a structure of formula II or anunmethylated cap with a structure of formula III.

wherein,R₂ is H or CH₃;R₄ is NH₂;R₅ is OH or OCH₃;R₆ is H or CH₃; andM is a nucleotide of the mRNA.

wherein M is a nucleotide of the mRNA.

In some embodiments, step (4) further separates methylated andunmethylated capped RNA. In some embodiments, the methylated capcomprises methylation at R₃ and/or R₅ position as shown in formula I. Insome embodiments, an inventive method according to the present inventionfurther includes a step of quantitatively determining methylationpercentage of the capped RNA.

In some embodiments, a suitable DNA oligonucleotide is about 10-80nucleotides in length (e.g., about 10-75, 10-70, 10-65, 10-60, 10-55,10-50, 10-45, 10-40, 10-35, 10-30, 10-25, 10-20, or 10-15 nucleotides inlength). In some embodiments, a suitable DNA oligonucleotide is orgreater than about 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70,75, or 80 nucleotides in length. In some embodiments, a suitable DNAoligonucleotide is flanked on one or both sides by one or more RNAnucleotides (e.g., by 1, 2, 3, 4, 5, or more RNA nucleotides).

In some embodiments, a suitable DNA oligonucleotide is complimentary toa sequence in the 5′ untranslated region of the mRNA within 1, 2, 3, 4,or 5 bases from the cap or uncapped penultimate base of mRNA.

In some embodiments, the one or more nucleases that selectively degradeDNA/RNA hybrid and/or unannealed RNA comprise RNase H. In someembodiments, the one or more nucleases that selectively degrade DNA/RNAhybrid and/or unannealed RNA comprise a nuclease that generatesblunt-ended capped and uncapped fragments. In some embodiments, the oneor more nucleases comprise nuclease S1 and/or RNAse H, or another 5′exonuclease.

In some embodiments, the capped and uncapped fragments from step (3)comprise no more than 5 bases (e.g., no more than 4, 3, 2, or 1) of themRNA. In some embodiments, the capped and uncapped fragments from step(3) comprise no more than 2 bases of the mRNA.

In some embodiments, step (4) of inventive methods described hereincomprises a step of applying the capped and uncapped fragments to achromatographic column. In some embodiments, a suitable chromatographiccolumn is selected from the group consisting of an anion-exchange HPLCcolumn, a cation-exchange HPLC column, a reverse phase HPLC column, ahydrophobic interaction column, an ultra-performance liquidchromatography column, or a size exclusion column.

In some embodiments, step (5) of inventive methods described hereincomprises determining relative peak areas of the capped and uncappedfragments.

In some embodiments, inventive methods according to the presentinvention are used to quantify mRNA capping efficiency of an mRNA samplesynthesized in vitro.

Among other things, the present invention further provides compositionsand kits for performing inventive methods described herein. In someembodiments, the present invention provides a kit of quantifying mRNAcapping efficiency, containing one or more of: (1) a DNA oligonucleotidecomplimentary to a sequence in the 5′ untranslated region of an mRNA tobe quantified adjacent to the cap or uncapped penultimate base of mRNA;(2) one or more reagents for annealing between DNA and RNA; (3) one ormore nucleases (e.g., RNAse H and/or nuclease S1) that selectivelydegrade DNA/RNA hybrid and/or unannealed mRNA; and (4) one or morereagents for performing chromatography (e.g., a chromatographic column).

In yet another aspect, the present invention provides methods ofmanufacturing mRNA comprising a step of quantifying mRNA cappingefficiency according to inventive methods described herein. In someembodiments, manufacturing methods according to the present inventioncontain a step of adjusting a manufacturing condition based on theresult from quantifying mRNA capping efficiency. In some embodiments,the quantifying step is conducted before releasing an mRNA lot.

Among other things, the present invention further provides mRNAmanufactured according to methods described herein.

As used in this application, the terms “about” and “approximately” areused as equivalents. Any numerals used in this application with orwithout about/approximately are meant to cover any normal fluctuationsappreciated by one of ordinary skill in the relevant art.

Other features, objects, and advantages of the present invention areapparent in the detailed description that follows. It should beunderstood, however, that the detailed description, while indicatingembodiments of the present invention, is given by way of illustrationonly, not limitation. Various changes and modifications within the scopeof the invention will become apparent to those skilled in the art fromthe detailed description.

BRIEF DESCRIPTION OF THE DRAWINGS

The drawings are for illustration purposes and are in no way limiting.

FIG. 1 depicts an exemplary embodiment comprising enzymatic manipulationof mRNA prior to chromatographic separation. A DNA oligonucleotideprimer is used to anneal the mRNA adjacent to Cap0 or Cap1 structures,if present. A nuclease is provided to digest the RNA in the DNA:RNAhybrid, thereby producing a capped or uncapped analyte.

FIG. 2 is a diagram of exemplary mRNA capped structures and an uncappedstructure present in various embodiments of the invention.

FIG. 3 is a bar graph demonstrating quantification of secreted humanalpha-galactosidase (GLA) protein levels as measured via ELISA. Theprotein detected is a result of its production from GLA mRNA deliveredintravenously via a single dose of lipid nanoparticles (30 ugencapsulated GLA mRNA) six hours post-administration.

DEFINITIONS

In order for the present invention to be more readily understood,certain terms are first defined. Additional definitions for thefollowing terms and other terms are set forth throughout thespecification.

Affinity: As is known in the art, “affinity” is a measure of thetightness with which a particular ligand binds to (e.g., associatesnon-covalently with) and/or the rate or frequency with which itdissociates from, its partner. As is known in the art, any of a varietyof technologies can be utilized to determine affinity. In manyembodiments, affinity represents a measure of specific binding.

Anneal or hybridization: As used herein, the terms “anneal,”“hybridization,” and grammatical equivalent, refers to the formation ofcomplexes (also called duplexes or hybrids) between nucleotide sequenceswhich are sufficiently complementary to form complexes via Watson-Crickbase pairing or non-canonical base pairing. It will be appreciated thatannealing or hybridizing sequences need not have perfect complementaryto provide stable hybrids. In many situations, stable hybrids will formwhere fewer than about 10% of the bases are mismatches. Accordingly, asused herein, the term “complementary” refers to a nucleic acid moleculethat forms a stable duplex with its complement under particularconditions, generally where there is about 90% or greater homology(e.g., about 95% or greater, about 98% or greater, or about 99% orgreater homology). Those skilled in the art understand how to estimateand adjust the stringency of hybridization conditions such thatsequences that have at least a desired level of complementarity willstably hybridize, while those having lower complementarity will not. Forexamples of hybridization conditions and parameters, see, for example,Sambrook et al., “Molecular Cloning: A Laboratory Manual”, 1989, SecondEdition, Cold Spring Harbor Press: Plainview, N.Y. and Ausubel, “CurrentProtocols in Molecular Biology”, 1994, John Wiley & Sons: Secaucus, N.J.Complementarity between two nucleic acid molecules is said to be“complete”, “total” or “perfect” if all the nucleic acid's bases arematched, and is said to be “partial” otherwise.

Approximately: As used herein, the term “approximately” or “about,” asapplied to one or more values of interest, refers to a value that issimilar to a stated reference value. In certain embodiments, the term“approximately” or “about” refers to a range of values that fall within25%, 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%,6%, 5%, 4%, 3%, 2%, 1%, or less in either direction (greater than orless than) of the stated reference value unless otherwise stated orotherwise evident from the context (except where such number wouldexceed 100% of a possible value).

Chromatography: As used herein, the term “chromatography” refers to atechnique for separation of mixtures. Typically, the mixture isdissolved in a fluid called the “mobile phase,” which carries it througha structure holding another material called the “stationary phase.”Column chromatography is a separation technique in which the stationarybed is within a tube, i.e., a column.

Compound and Agent: The terms “compound” and “agent” are used hereininterchangeably. They refer to any naturally occurring or non-naturallyoccurring (i.e., synthetic or recombinant) molecule, such as abiological macromolecule (e.g., nucleic acid, polypeptide or protein),organic or inorganic molecule, or an extract made from biologicalmaterials such as bacteria, plants, fungi, or animal (particularlymammalian, including human) cells or tissues. The compound may be asingle molecule or a mixture or complex of at least two molecules.

Control: As used herein, the term “control” has its art-understoodmeaning of being a standard against which results are compared.Typically, controls are used to augment integrity in experiments byisolating variables in order to make a conclusion about such variables.In some embodiments, a control is a reaction or assay that is performedsimultaneously with a test reaction or assay to provide a comparator. Inone experiment, the “test” (i.e., the variable being tested) is applied.In the second experiment, the “control,” the variable being tested isnot applied. In some embodiments, a control is a historical control(i.e., of a test or assay performed previously, or an amount or resultthat is previously known). In some embodiments, a control is orcomprises a printed or otherwise saved record. A control may be apositive control or a negative control.

Kit: As used herein, the term “kit” refers to any delivery system fordelivering materials. Such delivery systems may include systems thatallow for the storage, transport, or delivery of various diagnostic ortherapeutic reagents (e.g., oligonucleotides, antibodies, enzymes, etc.in the appropriate containers) and/or supporting materials (e.g.,buffers, written instructions for performing the assay etc.) from onelocation to another. For example, kits include one or more enclosures(e.g., boxes) containing the relevant reaction reagents and/orsupporting materials. As used herein, the term “fragmented kit” refersto delivery systems comprising two or more separate containers that eachcontains a subportion of the total kit components. The containers may bedelivered to the intended recipient together or separately. For example,a first container may contain an enzyme for use in an assay, while asecond container contains oligonucleotides. The term “fragmented kit” isintended to encompass kits containing Analyte Specific Reagents (ASR's)regulated under section 520(e) of the Federal Food, Drug, and CosmeticAct, but are not limited thereto. Indeed, any delivery system comprisingtwo or more separate containers that each contains a subportion of thetotal kit components are included in the term “fragmented kit.” Incontrast, a “combined kit” refers to a delivery system containing all ofthe components in a single container (e.g., in a single box housing eachof the desired components). The term “kit” includes both fragmented andcombined kits.

Nucleoside: The term “nucleoside” or “nucleobase”, as used herein,refers to adenine (“A”), guanine (“G”), cytosine (“C”), uracil (“U”),thymine (“T”) and analogs thereof linked to a carbohydrate, for exampleD-ribose (in RNA) or 2′-deoxy-D-ribose (in DNA), through an N-glycosidicbond between the anomeric carbon of the carbohydrate (1′-carbon atom ofthe carbohydrate) and the nucleobase. When the nucleobase is purine,e.g., A or G, the ribose sugar is generally attached to the N9-positionof the heterocyclic ring of the purine. When the nucleobase ispyrimidine, e.g., C, T or U, the sugar is generally attached to theN1-position of the heterocyclic ring. The carbohydrate may besubstituted or unsubstituted. Substituted ribose sugars include, but arenot limited to, those in which one or more of the carbon atoms, forexample the 2′-carbon atom, is substituted with one or more of the sameor different Cl, F, —R, —OR, —NR₂ or halogen groups, where each R isindependently H, C₁-C₆ alkyl or C₅-C₁₄ aryl. Ribose examples includeribose, 2′-deoxyribose, 2′,3′-dideoxyribose, 2′-haloribose,2′-fluororibose, 2′-chlororibose, and 2′-alkylribose, e.g., 2′-O-methyl,4′-alpha-anomeric nucleotides, l′-alpha-anomeric nucleotides (Asselineet al., NUCL. ACIDS RES., 19:4067-74 [1991]), 2′-4′- and 3′-4′-linkedand other “locked” or “LNA,” bicyclic sugar modifications (WO 98/22489;WO 98/39352; WO 99/14226).

Nucleotide: The term “nucleotide” as used herein means a nucleoside in aphosphorylated form (a phosphate ester of a nucleoside), as a monomerunit or within a polynucleotide polymer. “Nucleotide 5′-triphosphate”refers to a nucleotide with a triphosphate ester group at the 5′position, sometimes denoted as “NTP”, or “dNTP” and “ddNTP” toparticularly point out the structural features of the ribose sugar. Thetriphosphate ester group may include sulfur substitutions for thevarious oxygen moieties, e.g., alpha-thio-nucleotide 5′-triphosphates.Nucleotides can exist in the mono-, di-, or tri-phosphorylated forms.The carbon atoms of the ribose present in nucleotides are designatedwith a prime character (′) to distinguish them from the backbonenumbering in the bases. For a review of polynucleotide and nucleic acidchemistry see Shabarova, Z. and Bogdanov, A. Advanced Organic Chemistryof Nucleic Acids, VCH, New York, 1994.

Nucleic acid: The terms “nucleic acid”, “nucleic acid molecule”,“polynucleotide” or “oligonucleotide” may be used hereininterchangeably. They refer to polymers of nucleotide monomers oranalogs thereof, such as deoxyribonucleic acid (DNA) and ribonucleicacid (RNA) and combinations thereof. The nucleotides may be genomic,synthetic or semi-synthetic in origin. Unless otherwise stated, theterms encompass nucleic acid-like structures with synthetic backbones,as well as amplification products. As will be appreciated by one skilledin the art, the length of these polymers (i.e., the number ofnucleotides it contains) can vary widely, often depending on theirintended function or use. Polynucleotides can be linear, branchedlinear, or circular molecules. Polynucleotides also have associatedcounter ions, such as H⁺, NH₄ ⁺, trialkylammonium, Mg⁺, Na⁺ and thelike. A polynucleotide may be composed entirely of deoxyribonucleotides,entirely of ribonucleotides, or chimeric mixtures thereof.Polynucleotides may be composed of internucleotide nucleobase and sugaranalogs.

In some embodiments, the term “oligonucleotide” is used herein to denotea polynucleotide that comprises between about 5 and about 150nucleotides, e.g., between about 10 and about 100 nucleotides, betweenabout 15 and about 75 nucleotides, or between about 15 and about 50nucleotides. Throughout the specification, whenever an oligonucleotideis represented by a sequence of letters (chosen, for example, from thefour base letters: A, C, G, and T, which denote adenosine, cytidine,guanosine, and thymidine, respectively), the nucleotides are presentedin the 5′ to 3′ order from the left to the right. A “polynucleotidesequence” refers to the sequence of nucleotide monomers along thepolymer. Unless denoted otherwise, whenever a polynucleotide sequence isrepresented, it will be understood that the nucleotides are in 5′ to 3′orientation from left to right.

Nucleic acids, polynucleotides and oligonucleotides may be comprised ofstandard nucleotide bases or substituted with nucleotide isoformanalogs, including, but not limited to iso-C and iso-G bases, which mayhybridize more or less permissibly than standard bases, and which willpreferentially hybridize with complementary isoform analog bases. Manysuch isoform bases are described, for example, by Benner et al., (1987)Cold Spring Harb. Symp. Quant. Biol. 52, 53-63. Analogs of naturallyoccurring nucleotide monomers include, for example, 7-deazaadenine,7-deazaguanine, 7-deaza-8-azaguanine, 7-deaza-8-azaadenine,7-methylguanine, inosine, nebularine, nitropyrrole (Bergstrom, J. Amer.Chem. Soc., 117:1201-1209 [1995]), nitroindole, 2-aminopurine,2-amino-6-chloropurine, 2,6-diaminopurine, hypoxanthine, pseudouridine,pseudocytosine, pseudoisocytosine, 5-propynylcytosine, isocytosine,isoguanine (Seela, U.S. Pat. No. 6,147,199), 7-deazaguanine (Seela, U.S.Pat. No. 5,990,303), 2-azapurine (Seela, WO 01/16149), 2-thiopyrimidine,6-thioguanine, 4-thiothymine, 4-thiouracil, 0-6-methylguanine,N-6-methyladenine, O-4-methylthymine, 5,6-dihydrothymine,5,6-dihydrouracil, 4-methylindole, pyrazolo[3,4-D]pyrimidines, “PPG”(Meyer, U.S. Pat. Nos. 6,143,877 and 6,127,121; Gall, WO 01/38584), andethenoadenine (Fasman (1989) in Practical Handbook of Biochemistry andMolecular Biology, pp. 385-394, CRC Press, Boca Raton, Fla.).

The term “3′” refers to a region or position in a polynucleotide oroligonucleotide 3′ (i.e., downstream) from another region or position inthe same polynucleotide or oligonucleotide. The term “5′” refers to aregion or position in a polynucleotide or oligonucleotide 5′ (i.e.,upstream) from another region or position in the same polynucleotide oroligonucleotide. The terms “3′ end” and “3′ terminus”, as used herein inreference to a nucleic acid molecule, refer to the end of the nucleicacid which contains a free hydroxyl group attached to the 3′ carbon ofthe terminal pentose sugar. The term “5′ end” and “5′ terminus”, as usedherein in reference to a nucleic acid molecule, refers to the end of thenucleic acid molecule which contains a free hydroxyl or phosphate groupattached to the 5′ carbon of the terminal pentose sugar. In someembodiments of the invention, oligonucleotide primers comprise tracts ofpoly-adenosine at their 5′ termini.

Target: As used herein, the term “target” refers to a molecule ofinterest.

DETAILED DESCRIPTION

The present invention provides, among other things, improved methods forquantifying mRNA capping efficiency. In some embodiments, the presentinvention provides a method of quantifying mRNA capping efficiency basedon generating capped and uncapped fragments, separating the capped anduncapped fragments by chromatography, and determining relative amount ofthe capped and uncapped fragments. In some embodiments, the capped anduncapped fragments can be generated by contacting an mRNA sample with aDNA oligonucleotide complimentary to a sequence in the 5′ untranslatedregion of the mRNA adjacent to the cap or uncapped penultimate base ofmRNA under conditions that permit the DNA oligonucleotide to anneal tothe sequence; and selectively degrading DNA/RNA hybrid and/or unannealedmRNA by one or more nucleases (e.g., nuclease S1, RNAse H, and/or other5′ exonuclease). In some embodiments, a chromatography-based method canfurther separate methylated and unmethylated capped mRNA and determinemethylation state and percentage of the capped mRNA.

Various embodiments of the present invention are useful in quantitatingcapping efficiency during in vitro mRNA synthesis. Thus, the presentinvention provides an important quality control approach formanufacturing mRNA and, in particular, for assessing the safety,efficacy and commercially feasibility of mRNAs with therapeuticapplications.

Various aspects of the invention are described in detail in thefollowing sections. The use of sections is not meant to limit theinvention. Each section can apply to any aspect of the invention. Inthis application, the use of “or” means “and/or” unless statedotherwise.

mRNA Capping and/or Methylation

Typically, eukaryotic mRNAs bear a “cap” structure at their 5′-termini,which plays an important role in translation. For example, the cap playsa pivotal role in mRNA metabolism, and is required to varying degreesfor processing and maturation of an RNA transcript in the nucleus,transport of mRNA from the nucleus to the cytoplasm, mRNA stability, andefficient translation of the mRNA to protein. The 5′ cap structure isinvolved in the initiation of protein synthesis of eukaryotic cellularand eukaryotic viral mRNAs and in mRNA processing and stability in vivo(see, e.g, Shatkin, A. J., CELL, 9: 645-653 (1976); Furuichi, et al.,NATURE, 266: 235 (1977); FEDERATION OF EXPERIMENTAL BIOLOGISTS SOCIETYLETTER 96: 1-11 (1978); Sonenberg, N., PROG. NUC. ACID RES MOL BIOL, 35:173-207 (1988)). Specific cap binding proteins exist that are componentsof the machinery required for initiation of translation of an mRNA (see,e.g., Shatkin, A. J., CELL, 40: 223-24 (1985); Sonenberg, N., PROG. NUC.ACID RES MOL BIOL, 35: 173-207 (1988)). The cap of mRNA is recognized bythe translational initiation factor eIF4E (Gingras, et al., ANN. REV.BIOCHEM. 68: 913-963 (1999); Rhoads, R. E., J. BIOL. CHEM. 274:30337-3040 (1999)). The 5′ cap structure also provides resistance to5′-exonuclease activity and its absence results in rapid degradation ofthe mRNA (see, e.g., Ross, J., MOL. BIOL. MED. 5: 1-14 (1988); Green, M.R. et al., CELL, 32: 681-694 (1983)). Since the primary transcripts ofmany eukaryotic cellular genes and eukaryotic viral genes requireprocessing to remove intervening sequences (introns) within the codingregions of these transcripts, the benefit of the cap also extends tostabilization of such pre-mRNA.

In vitro, capped RNAs have been reported to be translated moreefficiently than uncapped transcripts in a variety of in vitrotranslation systems, such as rabbit reticulocyte lysate or wheat germtranslation systems (see, e.g., Shimotohno, K., et al., PROC. NATL.ACAD. SCI. USA, 74: 2734-2738 (1977); Paterson and Rosenberg, NATURE,279: 692 (1979)). This effect is also believed to be due in part toprotection of the RNA from exoribonucleases present in the in vitrotranslation system, as well as other factors.

Naturally occurring cap structures comprise a 7-methyl guanosine that islinked via a triphosphate bridge to the 5′-end of the first transcribednucleotide, resulting in a dinucleotide cap of m⁷G(5′)ppp(5′)N, where Nis any nucleoside. In vivo, the cap is added enzymatically. The cap isadded in the nucleus and is catalyzed by the enzyme guanylyltransferase. The addition of the cap to the 5′ terminal end of RNAoccurs immediately after initiation of transcription. The terminalnucleoside is typically a guanosine, and is in the reverse orientationto all the other nucleotides, i.e., G(5′)ppp(5′)GpNpNp.

A common cap for mRNA produced by in vitro transcription ism⁷G(5′)ppp(5′)G, which has been used as the dinucleotide cap intranscription with T7 or SP6 RNA polymerase in vitro to obtain RNAshaving a cap structure in their 5′-termini. The prevailing method forthe in vitro synthesis of capped mRNA employs a pre-formed dinucleotideof the form m⁷G(5′)ppp(5′)G (“m⁷GpppG”) as an initiator oftranscription. A disadvantage of using m⁷G(5′)ppp(5′)G, apseudosymmetrical dinucleotide, is the propensity of the 3′-OH of eitherthe G or m⁷G moiety to serve as the initiating nucleophile fortranscriptional elongation. In other words, the presence of a 3′-OH onboth the m⁷G and G moieties leads to up to half of the mRNAsincorporating caps in an improper orientation. This leads to thesynthesis of two isomeric RNAs of the form m⁷G(5′)pppG(pN)_(n) andG(5′)pppm⁷G(pN)n, in approximately equal proportions, depending upon theionic conditions of the transcription reaction. Variations in theisomeric forms can adversely effect in vitro translation and areundesirable for a homogenous therapeutic product.

To date, the usual form of a synthetic dinucleotide cap used in in vitrotranslation experiments is the Anti-Reverse Cap Analog (“ARCA”), whichis generally a modified cap analog in which the 2′ or 3′ OH group isreplaced with —OCH₃. ARCA and triple-methylated cap analogs areincorporated in the forward orientation. Chemical modification of m⁷G ateither the 2′ or 3′ OH group of the ribose ring results in the cap beingincorporated solely in the forward orientation, even though the 2′ OHgroup does not participate in the phosphodiester bond. (Jemielity, J. etal., “Novel ‘anti-reverse’ cap analogs with superior translationalproperties”, RNA, 9: 1108-1122 (2003)). The selective procedure formethylation of guanosine at N7 and 3′ O-methylation and 5′ diphosphatesynthesis has been established (Kore, A. and Parmar, G. NUCLEOSIDES,NUCLEOTIDES, AND NUCLEIC ACIDS, 25:337-340, (2006) and Kore, A. R., etal. NUCLEOSIDES, NUCLEOTIDES, AND NUCLEIC ACIDS 25(3): 307-14, (2006).

Transcription of RNA usually starts with a nucleoside triphosphate(usually a purine, A or G). In vitro transcription typically comprises aphage RNA polymerase such as T7, T3 or SP6, a DNA template containing aphage polymerase promoter, nucleotides (ATP, GTP, CTP and UTP) and abuffer containing magnesium salt. The synthesis of capped RNA includesthe incorporation of a cap analog (e.g., m⁷GpppG) in the transcriptionreaction, which in some embodiments is incorporated by the addition ofrecombinant guanylyl transferase. Excess m⁷GpppG to GTP (4:1) increasesthe opportunity that each transcript will have a 5′ cap. Kits forcapping of in vitro transcribed mRNAs are commercially available,including the mMESSAGE mMACHINE® kit (Ambion, Inc., Austin, Tex.). Thesekits will typically yield 80% capped RNA to 20% uncapped RNA, althoughtotal RNA yields are lower as GTP concentration becomes rate limiting asGTP is needed for the elongation of the transcript. However, currentlythere is no technology/method available that will allow quantificationof capping efficiency without permanent alterations of the mRNAs in asample.

Methods of estimating capping efficiency are known in the art. Forexample, the T7 RNA polymerase can be incubated with a cap dinucleotide,all four ribonucleotide triphosphates, [α-³²P]GTP, and a short DNAtemplate in which G is the first ribonucleotide specified after thepromoter (see Grudzien, E. et al. “Novel cap analogs for in vitrosynthesis of mRNA with high translation efficiency”, RNA, 10: 1479-1487(2004)). Any nucleotide on the 5′ side of a G residue acquires a³²P-labeled 3′-phosphate group after RNase T2 digestion bynearest-neighbor transfer. Anion exchange chromatography is then used toresolve labeled nucleoside 3′-monophosphates, resulting from internalpositions in the RNA, from 5′-terminal products. 5′-terminal productsare of two types. Uncapped RNAs yield labeled guanosine 5′-triphosphate3′-monophosphate (p3Gp*; in which * indicates the labeled phosphategroup). Capped RNAs yield various 5′-terminal structures, depending onthe nature of the cap analog used (m⁷Gp3Gp* and Gp3 m⁷Gp* when the capanalog is m⁷Gp3G).

However, a major drawback of these methods is that the entire sample isrendered radioactive or otherwise destroyed, and thus cannot be used insubsequent therapeutic applications. Although in theory a separatequantification reaction could be run alongside a therapeutic synthesisreaction, such arrangements are inadequate. Simultaneous but separatesamples are inherently variable due to intra-operator error and minutevariations in reaction conditions. This is particularly true forquantifications using a standard curve, in which a value for a point onthe standard curve on one given day may not be the same on the next day.To obtain accurate results in calculating capping efficiency, it isdesirable to use a representative sample taken from the therapeuticsynthesis reaction, a sample for which capping efficiency can beevaluated relative to controls and which is representative of thecapping efficiency in the therapeutic synthesis reaction.

Thus, the present invention provides improved methods of directlyquantitating mRNA capping efficiency in a sample (e.g., a representativealiquot sample from an in vitro synthesis reaction). Some embodiments ofthe invention comprise chromatographic methods of quantitating mRNAcapping efficiency. These methods are based in part on the insights thatthe versatility of enzymatic manipulation can be used to increase theresolution of chromatographic separation of polynucleotides. (FIG. 1)Thus, by amplifying the power of chromatographic separation throughenzymatic manipulation, embodiments of the invention increase theefficiency, quality and throughput of quantitation. For example, notonly can the chromatographic methods described herein quantitate cappingefficiency, they can also provide information on the modification of thecap (e.g., methylation status at particular cap positions). Thus,embodiments of the invention can simultaneously quantitate cappingefficiency and the efficiency of cap modification (e.g., methlylationefficiency). This quantification provides important characterization ofan mRNA drug product that has significant impact on the proteinproduction.

Chromatographic embodiments of the invention can be used to quantify anyof the cap structure and cap analogs described herein, as well asvarious modifications within the caps. Particular embodiments utilizethe natural biological activity of specific nucleases to providequantitative information of capping efficiency and the efficiency ofmethylation of the cap guanine base (e.g., N-7 position). Additionalembodiments can also simultaneously quantitate methylation of the 2′-Oposition of the ribose ring for the penultimate base (Cap1 structure,see FIG. 2). Embodiments of the invention may be used to quantify thecapping efficiency of a wide variety of RNA species, including in vitrotranscribed mRNA, isolated eukaryotic mRNA, and viral RNA.

Enzymatic manipulation is facilitated through the use of a DNAoligonucleotide complimentary to a sequence in the 5′ untranslatedregion of the mRNA adjacent to the cap or uncapped penultimate base ofmRNA. (FIG. 1) The DNA oligonucleotide is added to an mRNA samplecomprising capped mRNA and uncapped mRNA under conditions that permitthe DNA oligonucleotide to anneal to the specified sequence in theuntranslated region. A nuclease is then provided, which selectivelydegrades DNA/RNA hybrid and/or unannealed mRNA, resulting in capped anduncapped 5′ fragments. (FIG. 1) In some embodiments, at least a portionof the fragment is double-stranded. In some embodiments, thedouble-stranded portion is at least partially an RNA:RNA hybrid. In someembodiments, the double-stranded portion is at least partially anDNA:RNA hybrid. Fragments resulting from nuclease treatment may beblunt-ended or staggered. In some embodiments, the fragments are between2-20 nucleotides (including a cap nucleotide if present); i.e. fragmentsresulting from nuclease treatment can be 20 nucleotides, 19 nucleotides,18 nucleotides, 17 nucleotides, 16 nucleotides, 15 nucleotides, 14nucleotides, 13 nucleotides, 12 nucleotides, 11 nucleotides, 10nucleotides, 9 nucleotides, 8 nucleotides, 7 nucleotides, 6 nucleotides,5 nucleotides, 4 nucleotides, 3 nucleotides or 2 nucleotides. In someembodiments, the capped and uncapped fragments comprise no more than 5bases of the mRNA. In some embodiments, the capped and uncappedfragments comprise no more than 2 bases of the mRNA.

The capped and uncapped fragments are then resolved (i.e., separatedfrom one another) by chromatography. The amount of capped and uncappedfragments can then be quantitated by standard quantitativechromatography techniques, for example HPLC peak integration.

Embodiments of the invention are not limited by the type or size of DNAoligonucleotide. In some embodiments, the oligonucleotide comprisesbetween 10-80 nucleotides; e.g., 10 nucleotides, 15 nucleotides, 20nucleotides, 25 nucleotides, 30 nucleotides, 35 nucleotides, 40nucleotide, 45 nucleotides, 50 nucleotides or more. The size of the DNAoligonucleotide can be selected to generate a capped fragment (ifpresent) of desired length. The DNA oligonucleotide may also be designedto hybridize to any region of the 5′ untranslated region depending onwhere cleavage is desired; i.e., can be positioned within the 5′untranslated region to produce a capped fragment (if present) of anysize. In particular, a suitably designed oligonucleotide comprising asmall stretch of DNA bases (e.g. about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10,12, 14, 16, 18, or 20 nucleotides) flanked by RNA bases (e.g., 1-15) oneach side (i.e., a “gapmer”) can be annealed to the mRNA analyte.Designing such an oligonucleotide to bind to the complementary bases ofthe 5′ untranslated region of the mRNA allows for select cleavage viaDNA/RNA hybrid recognition of RNAse H. Similarly, a suitably designedoligonucleotide may comprise a small stretch of DNA bases (e.g., about1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 14, 16, 18 or 20 nucleotides) flankedonly at one end (e.g., the 5′ or 3′ end) by RNA bases (e.g., 1-15).

In preferred embodiments, the oligonucleotide is designed to binddirectly adjacent to the cap and/or penultimate base of the mRNAconstruct, allowing for the resulting cleaved bases to consist of theinitial two (or few) bases of the mRNA construct. Without wishing to bebound by any particular theory, it is thought desirable to produce smallcapped and uncapped fragments in order to improve the chromatographyresolution; i.e., to improve the separation of capped and uncappedfragments and quantify relative amounts. Generally speaking, the smallerthe fragment, the better the separation and quantification. In someembodiments, the first base of the DNA oligonucleotide binds at thepenultimate base of the mRNA. In some embodiments, the first base of theDNA oligonucleotide binds adjacent to the penultimate base of the mRNA(i.e., at M of Formula 1). In some embodiments, the first base of theDNA oligonucleotide binds at least 2 nucleotides, at least 3nucleotides, at least 4 nucleotides, at least 5 nucleotides, at least 6nucleotides, at least 7 nucleotides, at least 8 nucleotides, at least 9nucleotides or at least 10 nucleotides from the penultimate base of themRNA.

Embodiments of the invention are not limited by the identity of thenuclease. Any nuclease capable of cleaving or digesting at least onestrand of a DNA:RNA hybrid may be used. As mentioned above, multiplenucleases may be used in a single reaction to effect production of acapped fragment or produce both a capped fragment (if present) andblunt-end the capped fragment. In some embodiments, a suitable nucleaseis RNase H or an enzyme with RNAse H-like biochemical activity. RNases Hare a ubiquitous enzyme family that is divided into two distinctphylogenetic subtypes, Type 1 and Type 2, either of which may be used inparticular embodiments. The RNases H are unified by the common abilityto bind a single-stranded (ss) RNA that is hybridized to a complementaryDNA single strand, and then degrade the RNA portion of the RNA:DNAhybrid. While the RNases H have been implicated in DNA replication andrecombination, and repair, their physiological roles are not completelyunderstood. In vitro, the enzymes will also bind double-stranded (ds)DNA, ssDNA, ssRNA, and dsRNA, albeit with lower affinities than theybind to RNA:DNA hybrids. Due to the ubiquity of the enzyme, there areseveral sequences for RNase H known in the literature, each of whichvary somewhat in their amino acid sequences. U.S. Pat. No. 5,268,289discloses a thermostable RNase H, as does U.S. Pat. No. 5,500,370. U.S.Pat. No. 6,376,661 discloses a human RNase H and compositions and usesthereof. U.S. Pat. No. 6,001,652 discloses a human type 2 RNase H. U.S.Pat. No. 6,071,734 discloses RNase H from HBV polymerase. All of theseRNases H may be used in one more embodiments of the invention.

In some embodiments, a suitable nuclease is S1 nuclease. In someembodiments, multiple nucleases are used; for example RNase H and an S1nuclease. Additional nucleases that may be utilized, either alone or incombination, in embodiments of the invention include Benzonase®,Nuclease P1, Phosphodiesterase II, RNase A, and RNase T1. Someembodiments further comprise addition of a single-stranded DNA nucleaseto finally produce or modify the fragment. In some embodiments, it maybe desired to heat the sample (e.g., to about 60° C.) or apply thesample to a heated chromatographic column in order to effectuateproduction of the capped fragments.

The nuclease-treated sample is then applied to a chromatographic columnto separate capped from uncapped fragments. In addition to separatingcapped from uncapped fragment, chromatography can resolve and quantitatea methylated cap from an unmethylated guanine cap. In some embodiments,a methylated penultimate base (2′-O-methylated base) can be separated(resolved) and quantitated from a cap unmethylated at that position.Current chromatography protocols can differentiate as any combination ofthe species that may arise from the in vitro synthetic process. Variousaspects of chromatographic embodiments are discussed in more detailbelow.

mRNA Caps

Inventive methods described herein are generally amenable toquantification of any type of mRNA cap. In some embodiments, the cap hasa structure of formula I:

wherein B is a nucleobase, R₁ is selected from a halogen, OH, and OCH₃,R₂ is selected from H, OH, and OCH₃, R₃ is CH₃, CH₂CH₃, CH₂CH₂CH₃ orvoid, R₄ is NH₂, R₅ is selected from OH, OCH₃ and a halogen, n is 1, 2,or 3, and M is a nucleotide, i.e., the third base of mRNA. In particularembodiments, B is guanine, but can be any nucleobase. In someembodiments, the cap is m⁷G(5′)ppp(5′)G in which a 2′-O-methyl residueis present at the 2′ OH group of the ribose ring of base 1 (i.e., at theR₅ position of Formula 1).

Cap analogs may be or comprise any modified “G” base (e.g., one or moremodified guanine nucleotides). Suitable cap analogs include, but are notlimited to, a chemical structures selected from the group consisting ofm⁷GpppG, m⁷GpppA, m⁷GpppC; unmethylated cap analogs (e.g., GpppG);dimethylated cap analog (e.g., m^(2,7)GpppG), trimethylated cap analog(e.g., m^(2,2,7)GpppG), dimethylated symmetrical cap analogs (e.g.,m⁷Gpppm⁷G), or anti reverse cap analogs (e.g., ARCA; m^(7,2′Ome)GpppG,m^(7,2′d)GpppG, m^(7,3′Ome)GpppG, m^(7,3′d)GpppG and theirtetraphosphate derivatives) (see, e.g., Jemielity, J. et al., “Novel‘anti-reverse’ cap analogs with superior translational properties”, RNA,9: 1108-1122 (2003)).

In a preferred embodiment, the cap is a 7-methyl guanylate (“m⁷G”)linked via a triphosphate bridge to the 5′-end of the first transcribednucleotide, resulting in m⁷G(5′)ppp(5′)N, where N is any nucleoside. Apreferred embodiment of a m⁷G cap utilized in embodiments of theinvention is m⁷G(5′)ppp(5′)G.

In some embodiments, mRNA is uncapped. (FIG. 2A) Uncapped mRNA may bepresent in a sample (i.e., as a result of incomplete capping in an invitro transcription reaction) and/or may be used a control toquantitative the level of uncapped species in a sample. In someembodiments, the cap is a Cap0 structure. (FIG. 2B). Cap0 structureslack a 2′-O-methyl residue of the ribose attached to bases 1 and 2. Insome embodiments, the cap is a Cap1 structure. (FIG. 2C) Cap1 structureshave a 2′-O-methyl residue at base 1. In some embodiments, the cap is aCap2 structure. Cap2 structures have a 2′-O-methyl residue attached toboth bases 1 and 2.

A variety of m⁷G cap analogs are known in the art, many of which arecommercially available. These include the m⁷GpppG described above, aswell as the ARCA 3′-OCH₃ and 2′-OCH₃ cap analogs (Jemielity, J. et al.,RNA, 9: 1108-1122 (2003)). Additional cap analogs for use in embodimentsof the invention include N7-benzylated dinucleoside tetraphosphateanalogs (described in Grudzien, E. et al., RNA, 10: 1479-1487 (2004)),phosphorothioate cap analogs (described in Grudzien-Nogalska, E., etal., RNA, 13: 1745-1755 (2007)), and cap analogs (including biotinylatedcap analogs) described in U.S. Pat. Nos. 8,093,367 and 8,304,529,incorporated by reference herein.

Production of Capped mRNAs

Capped mRNAs suitable for the quantitative methods disclosed herein maybe produced by any method known in the art.

In some embodiments, capped mRNA is produced by in vitro transcription,originally developed by Krieg and Melton (METHODS ENZYMOL., 1987, 155:397-415) for the synthesis of RNA using an RNA phage polymerase.Typically these reactions include at least a phage RNA polymerase (T7,T3 or SP6), a DNA template containing a phage polymerase promoter,nucleotides (ATP, CTP, GTP and UTP), and a buffer containing a magnesiumsalt. RNA synthesis yields may be optimized by increasing nucleotideconcentrations, adjusting magnesium concentrations and by includinginorganic pyrophosphatase (U.S. Pat. No. 5,256,555; Gurevich, et al.,ANAL. BIOCHEM. 195: 207-213 (1991); Sampson, J. R. and Uhlenbeck, O. C.,PROC. NATL. ACAD. SCI. USA. 85, 1033-1037 (1988); Wyatt, J. R., et al.,BIOTECHNIQUES, 11: 764-769 (1991)). Some embodiments utilize commercialkits for the large-scale synthesis of in vitro transcripts (e.g.,MEGAscript®, Ambion). The RNA synthesized in these reactions is usuallycharacterized by a 5′ terminal nucleotide that has a triphosphate at the5′ position of the ribose. Typically, depending on the RNA polymeraseand promoter combination used, this nucleotide is a guanosine, althoughit can be an adenosine (see e.g., Coleman, T. M., et al., NUCLEIC ACIDSRES., 32: e14 (2004)). In these reactions, all four nucleotides aretypically included at equimolar concentrations and none of them islimiting.

Some embodiment of the invention are batch reactions—that is, allcomponents are combined and then incubated at about 37° C. to promotethe polymerization of the RNA until the reaction terminates. Typically,a batch reaction is used for convenience and to obtain as much RNA asneeded from such reactions for their experiments. In some embodiments, a“fed-batch” system (see, e.g., JEFFREY A. KERN, BATCH AND FED-BATCHSTRATEGIES FOR LARGE-SCALE PRODUCTION OF RNA BY IN VITRO TRANSACTION(University of Colorado) (1997)) is used to increase the efficiency ofthe in vitro transcription reaction. All components are combined, butthen additional amounts of some of the reagents are added over time,such as the nucleotides and magnesium, to try to maintain constantreaction conditions. In addition, in some embodiments, the pH of thereaction may be held at 7.4 by monitoring it over time and adding KOH asneeded.

To synthesize a capped RNA by in vitro transcription, a cap analog(e.g., N-7 methyl GpppG; i.e., m⁷GpppG) is included in the transcriptionreaction. In some embodiments, the RNA polymerase will incorporate thecap analog as readily as any of the other nucleotides; that is, there isno bias for the cap analog. In some embodiments, the cap analog will beincorporated at the 5′ terminus by the enzyme guanylyl transferase Insome embodiments, the cap analog will be incorporated only at the 5′terminus because it does not have a 5′ triphosphate. In some embodimentsusing a T7, T3 and SP6 RNA polymerase, the +1 nucleotide of theirrespective promoters is usually a G residue and if both GTP and m⁷GpppGare present in equal concentrations in the transcription reaction, thenthey each have an equal chance of being incorporated at the +1 position.In some embodiments, m⁷GpppG is present in these reactions atseveral-fold higher concentrations than the GTP to increase the chancesthat a transcript will have a 5′ cap. In some embodiments, a mMESSAGEmMACHINE® kit (Cat. #1344, Ambion, Inc.) is used according tomanufacturer's instructions, where it is recommended that the cap to GTPratio be 4:1 (6 mM: 1.5 mM). In some embodiments, as the ratio of thecap analog to GTP increases in the reaction, the ratio of capped touncapped RNA increases proportionally. Considerations of cappingefficiency must be balanced with considerations of yield. Increasing theratio of cap analog to GTP in the transcription reaction produces loweryields of total RNA because the concentration of GTP becomes limitingwhen holding the total concentration of cap and GTP constant. Thus, thefinal RNA yield is dependent on GTP concentration, which is necessaryfor the elongation of the transcript. The other nucleotides (ATP, CTP,UTP) are present in excess.

In particular embodiments, mRNA are synthesized by in vitrotranscription from a plasmid DNA template encoding a gene of choice. Insome embodiments, in vitro transcription includes addition of a 5′ capstructure, Cap1 (FIG. 2C), which has a 2′-O-methyl residue at the 2′ OHgroup of the ribose ring of base 1, by enzymatic conjugation of GTP viaguanylyl transferase. In some embodiments, in vitro transcriptionincludes addition of a 5′ cap structure, Cap0 (FIG. 2B), which lacks the2′-O-methyl residue, by enzymatic conjugation of GTP via guanylyltransferase. In some embodiments, in vitro transcription includesaddition of a 5′ cap of any of the cap structures disclosed herein byenzymatic conjugation of GTP via guanylyl transferase. In someembodiments, a 3′ poly(A) tail of approximately 200 nucleotides inlength (as determined by gel electrophoresis) was incorporated throughthe addition of ATP in conjunction with PolyA polymerase. In someembodiments, the poly(A) tail is approximately 100-250 nucleotides inlength. In some embodiments, the poly(A) tail is about 50-300nucleotides in length. In some embodiments, the in vitro transcriptionproducts include 5′ and 3′ untranslated regions.

Chromatographic Separation

Embodiments of the invention utilize chromatography to provide highlyresolved (e.g. single base resolution) capped and uncapped fragments.Fragments can be efficiently resolved by thin-layer chromatography(“TLC”) or high performance liquid chromatography (“HPLC”). In thecontext of the present invention, the term “HPLC” includes various HPLCmethods as well as low or normal pressure liquid chromatography methods,which may be used to carry out some embodiments of the invention. Insome embodiments, fragments may be resolved by one or more of sizeexclusion chromatography-high performance liquid chromatography(SEC-HPLC) and/or reverse phase-high performance liquid chromatography(RP-HPLC) (e.g., using columns of octadecyl (C18)-bonded silica, andcarried out at an acidic pH with TFA as a counter-ion). In someembodiments of the invention, the major peak in the chromatogram iscapped mRNA fragments. Parameters that may be altered or optimized toincrease resolution include gradient conditions, organic modifier,counter ion, temperature, column pore size and particle size, solventcomposition and flow rate.

The quantitative methods described herein can include one or more stepsof ion exchange chromatography-HPLC (e.g., anion exchange-HPLC and/orcation exchange-HPLC). As will be known by those skilled in the art, ionexchangers (e.g., anion exchangers and/or cation exchangers) may bebased on various materials with respect to the matrix as well as to theattached charged groups. For example, the following matrices may beused, in which the materials mentioned may be more or less crosslinked:agarose based (such as Sepharose™ CL-6B, Sepharose™ Fast Flow andSepharose™ High Performance), cellulose based (such as DEAE Sephacel®),dextran based (such as SEPHADEX®), silica based and synthetic polymerbased.

The ion exchange resin can be prepared according to known methods.Typically, an equilibration buffer, which allows the resin to bind itscounter ions, can be passed through the ion exchange resin prior toloading the sample or composition comprising the polypeptide and one ormore contaminants onto the resin. Conveniently, the equilibration buffercan be the same as the loading buffer, but this is not required. In anoptional embodiment of the invention, the ion exchange resin can beregenerated with a regeneration buffer after elution of the polypeptide,such that the column can be re-used. Generally, the salt concentrationand/or pH of the regeneration buffer can be such that substantially allcontaminants and the polypeptide of interest are eluted from the ionexchange resin. Generally, the regeneration buffer has a very high saltconcentration for eluting contaminants and fragments from the ionexchange resin.

Some embodiments of the invention include, for example, subjectingsamples to anion exchange chromatography. High-resolution analysis ofnucleotide fragment may be performed as described in Ausser, W. A., etal., “High-resolution analysis and purification of syntheticoligonucleotides with strong anion-exchange HPLC”, BIOTECHNIQUES, 19:136-139 (1995), incorporated herein by reference. For the anion exchangeresin, the charged groups which are covalently attached to the matrixcan be, for example, diethylaminoethyl (DEAE), quaternary aminoethyl(QAE), and/or quaternary ammonium (Q). In some embodiments, the anionexchange resin employed is a Q Sepharose column. The anion exchangechromatography can be performed, for example, using, e.g., Q Sepharose™Fast Flow, Q Sepharose™ High Performance, Q Sepharose™ XL, Capto™ Q,DEAE, TOYOPEARL Gigacap® Q, Fractogel® TMAE (trimethylaminoethyl, aquarternary ammonia resin), Eshmuno™ Q, Nuvia™ Q, or UNOsphere™ Q. Otheranion exchangers can be used within the scope of the invention,including but not limited to, quaternary amine resins or “Q-resins”(e.g., Capto™-Q, Q-Sepharose®, QAE Sephadex®); diethylaminoethane resins(e.g., DEAE-Trisacryl®, DEAE Sepharose®, benzoylated naphthoylated DEAE,diethylaminoethyl Sephacel®); Amberjet® resins; Amberlyst® resins;Amberlite® resins (e.g., Amberlite® IRA-67, Amberlite® strongly basic,Amberlite® weakly basic), cholestyramine resin, ProPac^(@) resins (e.g.,ProPac^(@) SAX-10, ProPac^(@) WAX-10, ProPac® WCX-10); TSK-GEL® resins(e.g., TSKgel DEAE-NPR; TSKgel DEAE-5PW); and Acclaim® resins.

Typical mobile phases for anionic exchange chromatography includerelatively polar solutions, such as water, acetonitrile, organicalcohols such as methanol, ethanol, and isopropanol, or solutionscontaining 2-(N-morpholino)-ethanesulfonic acid (MES). Thus, in certainembodiments, the mobile phase includes about 0%, 1%, 2%, 4%, 6%, 8%,10%, 12%, 14%, 16%, 18%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%,65%, 70%, 75%, 80%, 85%, 90%, 95%, or about 100% polar solution. Incertain embodiments, the mobile phase comprises between about 1% toabout 100%, about 5% to about 95%, about 10% to about 90%, about 20% toabout 80%, about 30% to about 70%, or about 40% to about 60% polarsolution at any given time during the course of the separation.

In some embodiments, samples are subject to cation exchangechromatography, e.g., sulfopropyl (SP) cation exchange chromatography.In a typical embodiment, the cation exchange chromatography comprisessulfopropyl (SP) cation exchange chromatography, but other cationchromatography membranes or resins can be used, for example, a MUSTANG™S membrane, an S-Sepharose™ resin, or a Blue Sepharose™ resin. Thecation exchange chromatography can be performed at an optimizedtemperature to enhance target binding and/or decrease impurity binding.

In some embodiments, samples are subjected to hydrophobic interactionchromatography (HIC). Hydrophobic interaction chromatography utilizesthe attraction of a given molecule for a polar or non-polar environment,and in terms of nucleic acids, this propensity is governed by thehydrophobicity or hydrophilicity of nucleotides and modificationsthereon. Thus, nucleic acids are fractionated based upon their varyingdegrees of attraction to a hydrophobic matrix, typically an inertsupport with alkyl linker arms of 2-18 carbons in chain length. Thestationary phase consists of small non-polar groups (butyl, octyl, orphenyl) attached to a hydrophilic polymer backbone (e.g., cross-linkedSepharose™, dextran, or agarose). In some embodiments, the hydrophobicinteraction chromatography includes phenyl chromatography. In otherembodiments, the hydrophobic interaction chromatography includes butylchromatography or octyl chromatography.

In some embodiments, fragments are resolved by reverse phase-HPLC.Reversed phase HPLC consists of a non-polar stationary phase and amoderately polar mobile phase. In some embodiments, the stationary phaseis a silica which has been treated with, for example, RMe₂SiCl, where Ris a straight chain alkyl group such as C₁₈H₃₇ or C₈H₁₇. The retentiontime is therefore longer for molecules which are more non-polar innature, allowing polar molecules to elute more readily. Retention timeis increased by the addition of polar solvent to the mobile phase anddecreased by the addition of more hydrophobic solvent. Thecharacteristics of the specific RNA molecule as an analyte may play animportant role in its retention characteristics. In general, an analytehaving more non-polar functional groups (e.g., methyl groups) results ina longer retention time because it increases the molecule'shydrophobicity. Protocols for high resolution of RNA species usingreverse phase-HPLC, which may be adapted for use in embodiments of theinvention, are known in the art (see, e.g., U.S. pre-grant publication2010/0048883; Gilar, M., “Analysis and purification of syntheticoligonucleotides by reversed-phase high-performance liquidchromatography with photodiode array and mass spectrometry detection”,ANAL. BIOCHEM., 298: 196-206 (2001)).

Particular embodiments of the invention utilize combinations of thevarious chromatographic separations disclosed herein. For example,particular embodiments of the invention may utilize reverse-phaseion-pair chromatography, whereby separations are based on bothhydrophobicity and on the number of anions associated with the molecule,which may be used to purify fragments in a single HPLC step. Matriciescan be silica-based (e.g., Murray et al., ANAL. BIOCHEM., 218:177-184(1994)). Non-porous, inert polymer resins may be used in particularembodiments (see, e.g., Huber, C. G., “High-resolution liquidchromatography of oligonucleotides on nonporous alkylatedstyrene-divinylbenzene copolymers”, ANAL. BIOCHEM., 212: 351-358(1993)). Other combinations may be equally effective and must beevaluated in terms of the size of the fragment and the modificationssought to be resolved.

In some embodiments, a capping efficiency profile and/or methylationprofile may be determined by strong anion exchange chromatography usinga HPLC system. In general, uncapped mRNA adsorbs onto the fixed positivecharge of a strong anion exchange column and a gradient of increasingionic strength using a mobile phase at a predetermined flow rate elutescapped species (the cap bearing a positive charge) from the column inproportion to the strength of their ionic interaction with thepositively charged column. More negatively charged (more acidic)uncapped species elute later than less negatively charged (less acidic)capped species.

In certain embodiments, capped fragments are characterized by themethylation profile associated with the fragment. Typically, methylationprofiles reflect and quantitate the efficiency of methylation of the capguanine base (N-7 position). Additional embodiments can alsosimultaneously quantitate methylation of the 2′-O position of the ribosering for the penultimate base (Cap1 structure). In some embodiments, amethylation profile may be determined by performing reverse phase-HPLC,alone or in combination with ion exchange chromatography. In someembodiments, a “methylation profile” refers to a set of valuesrepresenting the amount of methylated capped fragment that elutes from acolumn at a point in time after addition to the column of a mobilephase. As described above, the retention time for methylated caps andpenultimate nucleotides, which are more non-polar in nature, isincreased relative to polar molecules, which elute more readily.Retention time may be increased by the addition of polar solvent to themobile phase and decreased by the addition of more hydrophobic solvent.

In some embodiments, ultra performance liquid chromatography (“UPLC”) isused to resolve capped and uncapped fragments, and optionally to provideadditional quantitative information on methylation states. UPLC refersgenerally to HPLC techniques using resin particle sizes less than 2.5μm, which provides a significant gain in efficiency even at increasedflow rates and linear velocities. By using small particles, speed andpeak capacity (number of peaks resolved per unit time) can be extended.Such techniques utilize chromatographic principles to run separationsusing columns packed with smaller particles and/or higher flow rates forincreased speed, with superior resolution and sensitivity. (see, e.g.,Swartz, M. E., “Ultra Performance Liquid Chromatography (UPLC): anintroduction”, SEPARATION SCIENCE REDEFINED (2005).)

In certain embodiments, chromatographic resolution (e.g., HPLC) may becombined with mass spectrometry (“MS”). LC-MS methods are known in theart for oligonucleotide separation and identification using aqueoustriethylammonium-hexafluoroisopropylalcohol (TEA HFIP) bufferscompatible with MS detection (Apffel, A., et al., “New procedure for theuse of HPLC-ESI MS for the analysis of nucleotides andoligonucleotides”, J. CHROMATOGR. A, 777: 3-21 (1997)). Alternatively, atriethylammonium bicarbonate mobile phase may be used foroligonucleotide separation with postcolumn acetonitrile addition to theeluent. The ion-pairing buffer may be chosen to give the best MSdetection sensitivity.

Quantitative analysis of capped fragment may also be performed usingreverse phase-HPLC columns packed with 2.5 μm fully porous C18 sorbent,as described in Gilar, M., ANAL. BIOCHEM., 298: 196-206 (2001).Parameters that may be optimized to enhance oligonucleotide masstransfer in the stationary phase include elevated temperature, smallsorbent particle size, and slow mobile phase flow rate. Atriethylammonium acetate (TEAA) buffer with UV detection and anoptimized TEA-HFIP mobile phase may be used for LC-MS separation andcharacterization of capped fragments.

In some embodiments, quantitation of capped fragments and themethylation status thereof, is achieved by automated integration ofrespective peak area in the HPLC chromatogram. Data may be presented asarea percent value, which refers to the percentage of a particularspecies' integrated peak area relative to the total integrated peak areaof the entire chromatograph.

In some embodiments, quantitation of capped and uncapped fragments maybe achieved through other appropriate methods, for example, massspectroscopy (MS)-based detection. It is contemplated that one of skillin the art may recognize additional applicable methods of quantitatingcapped and uncapped fragments as described herein.

Kits

The present invention further provides kits comprising various reagentsand materials useful for carrying out inventive methods according to thepresent invention. The quantitative procedures described herein may beperformed by diagnostic laboratories, experimental laboratories, orcommercial laboratories. The invention provides kits which can be usedin these different settings.

For example, materials and reagents for quantifying mRNA cappingefficiency in an mRNA sample by enzymatic manipulation andchromatographic separation may be assembled together in a kit. Incertain embodiments, an inventive kit comprises chromatographic columnsand optionally agents for separating capped mRNA fragments on thecolumn, and instructions for using the kit according to a method of theinvention.

Each kit may preferably comprise the reagent which renders the procedurespecific. Thus, for detecting/quantifying mRNA capping efficiency, kitsmay comprise a nucleic acid reagent of designed sequence thatspecifically anneals adjacent to an mRNA cap of a target. Kits may alsocomprise nucleases for production of the capped fragments; e.g., RNase Hand/or S1 nuclease. Kits may further include in vitro transcription andcapping reagents, enzymes and instructions for using the same.

Kits or other articles of manufacture according to the invention mayinclude one or more containers to hold various reagents. Suitablecontainers include, for example, bottles, vials, syringes (e.g.,pre-filled syringes), ampules. The container may be formed from avariety of materials such as glass or plastic.

In some embodiments, kits of the present invention may include suitablecontrol levels or control samples for determining control levels asdescribed herein. In some embodiments, kits of the invention may includeinstructions for using the kit according to one or more methods of theinvention and may comprise instructions for in vitro transcription andcapping.

EXAMPLES Example 1: Synthesis of mRNA

Firefly Luciferase (FFL) and human erythropoietin (EPO) mRNA weresynthesized by in vitro transcription from a plasmid DNA templateencoding each respective gene. In vitro transcription included additionof a 5′ cap structure, Cap1, which has a 2′-O-methyl residue at the 2′OH group of the ribose ring of base 1, by enzymatic conjugation of GTPvia guanylyl transferase. A 3′ poly(A) tail of approximately 200nucleotides in length (as determined by gel electrophoresis) wasincorporated through the addition of ATP in conjunction with PolyApolymerase (see detailed reaction conditions below). The in vitrotranscription product included 5′ and 3′ untranslated regions, which arerepresented as X and Y, respectively, in the sequences below:

Human Erythropoietin (EPO) mRNA (SEQ ID NO: 1)

X ₁AUGGGGGUGCACGAAUGUCCUGCCUGGCUGUGGCUUCUCCUGUCCCUGCUGUCGCUCCCUCUGGGCCUCCCAGUCCUGGGCGCCCCACCACGCCUCAUCUGUGACAGCCGAGUCCUGGAGAGGUACCUCUUGGAGGCCAAGGAGGCCGAGAAUAUCACGACGGGCUGUGCUGAACACUGCAGCUUGAAUGAGAAUAUCACUGUCCCAGACACCAAAGUUAAUUUCUAUGCCUGGAAGAGGAUGGAGGUCGGGCAGCAGGCCGUAGAAGUCUGGCAGGGCCUGGCCCUGCUGUCGGAAGCUGUCCUGCGGGGCCAGGCCCUGUUGGUCAACUCUUCCCAGCCGUGGGAGCCCCUGCAGCUGCAUGUGGAUAAAGCCGUCAGUGGCCUUCGCAGCCUCACCACUCUGCUUCGGGCUCUGGGAGCCCAGAAGGAAGCCAUCUCCCCUCCAGAUGCGGCCUCAGCUGCUCCACUCCGAACAAUCACUGCUGACACUUUCCGCAAACUCUUCCGAGUCUACUCCAAUUUCCUCCGGGGAAAGCUGAAGCUGUACACAGGGGAGGCCUGCAGGACAGGGGACAGAUGAY ₁

Codon-Optimized Firefly Luciferase (FFL) mRNA (SEQ ID NO: 2)

X ₂AUGGAAGAUGCCAAAAACAUUAAGAAGGGCCCAGCGCCAUUCUACCCACUCGAAGACGGGACCGCCGGCGAGCAGCUGCACAAAGCCAUGAAGCGCUACGCCCUGGUGCCCGGCACCAUCGCCUUUACCGACGCACAUAUCGAGGUGGACAUUACCUACGCCGAGUACUUCGAGAUGAGCGUUCGGCUGGCAGAAGCUAUGAAGCGCUAUGGGCUGAAUACAAACCAUCGGAUCGUGGUGUGCAGCGAGAAUAGCUUGCAGUUCUUCAUGCCCGUGUUGGGUGCCCUGUUCAUCGGUGUGGCUGUGGCCCCAGCUAACGACAUCUACAACGAGCGCGAGCUGCUGAACAGCAUGGGCAUCAGCCAGCCCACCGUCGUAUUCGUGAGCAAGAAAGGGCUGCAAAAGAUCCUCAACGUGCAAAAGAAGCUACCGAUCAUACAAAAGAUCAUCAUCAUGGAUAGCAAGACCGACUACCAGGGCUUCCAAAGCAUGUACACCUUCGUGACUUCCCAUUUGCCACCCGGCUUCAACGAGUACGACUUCGUGCCCGAGAGCUUCGACCGGGACAAAACCAUCGCCCUGAUCAUGAACAGUAGUGGCAGUACCGGAUUGCCCAAGGGCGUAGCCCUACCGCACCGCACCGCUUGUGUCCGAUUCAGUCAUGCCCGCGACCCCAUCUUCGGCAACCAGAUCAUCCCCGACACCGCUAUCCUCAGCGUGGUGCCAUUUCACCACGGCUUCGGCAUGUUCACCACGCUGGGCUACUUGAUCUGCGGCUUUCGGGUCGUGCUCAUGUACCGCUUCGAGGAGGAGCUAUUCUUGCGCAGCUUGCAAGACUAUAAGAUUCAAUCUGCCCUGCUGGUGCCCACACUAUUUAGCUUCUUCGCUAAGAGCACUCUCAUCGACAAGUACGACCUAAGCAACUUGCACGAGAUCGCCAGCGGCGGGGCGCCGCUCAGCAAGGAGGUAGGUGAGGCCGUGGCCAAACGCUUCCACCUACCAGGCAUCCGCCAGGGCUACGGCCUGACAGAAACAACCAGCGCCAUUCUGAUCACCCCCGAAGGGGACGACAAGCCUGGCGCAGUAGGCAAGGUGGUGCCCUUCUUCGAGGCUAAGGUGGUGGACUUGGACACCGGUAAGACACUGGGUGUGAACCAGCGCGGCGAGCUGUGCGUCCGUGGCCCCAUGAUCAUGAGCGGCUACGUUAACAACCCCGAGGCUACAAACGCUCUCAUCGACAAGGACGGCUGGCUGCACAGCGGCGACAUCGCCUACUGGGACGAGGACGAGCACUUCUUCAUCGUGGACCGGCUGAAGAGCCUGAUCAAAUACAAGGGCUACCAGGUAGCCCCAGCCGAACUGGAGAGCAUCCUGCUGCAACACCCCAACAUCUUCGACGCCGGGGUCGCCGGCCUGCCCGACGACGAUGCCGGCGAGCUGCCCGCCGCAGUCGUCGUGCUGGAACACGGUAAAACCAUGACCGAGAAGGAGAUCGUGGACUAUGUGGCCAGCCAGGUUACAACCGCCAAGAAGCUGCGCGGUGGUGUUGUGUUCGUGGACGAGGUGCCUAAAGGACUGACCGGCAAGUUGGACGCCCGCAAGAUCCGCGAGAUUCUCAUUAAGGCCAAGAAGGGCGGCAAGAUCGCCGUGUAY ₂

The 5′ and 3′ UTR sequences for X1/Y1 and X2/Y2 were as follows:

(SEQ ID NO: 3) X₁ = GGACAGAUCGCCUGGAGACGCCAUCCACGCUGUUUUGACCUCCAUAGAAGACACCGGGACCGAUCCAGCCUCCGCGGCCGGGAACGGUGCAUUGGAACGCGGAUUCCCCGUGCCAAGAGUGACUCACCGUCCUUGACACG  (SEQ ID NO: 4) X₂ =GGGAUCCUACC  (SEQ ID NO: 5) Y₁ =CGGGUGGCAUCCCUGUGACCCCUCCCCAGUGCCUCUCCUGGCCCUGGAAGUUGCCACUCCAGUGCCCACCAGCCUUGUCCUAAUAAAAUUAA GUUGCAUC (SEQ ID NO: 6) Y₂ = UUUGAAUU

The synthesis of mRNA was conducted under complete RNAse-freeconditions. All tubes, vials, pipette tips, pipettes, buffers, etc. wererequired nuclease-free. Messenger RNA was synthesized from a linearizedDNA template. To produce the desired mRNA pre-cursor (IVT) construct, amixture of ˜100 ug of linearized DNA, rNTPs (3.33 mM), DTT (10 mM), T7RNA polymerase, RNAse Inhibitor, Pyrophosphatase and reaction buffer(10×, 800 mM Hepes (pH8.0), 20 mM Spermidine, 250 mM MgCl₂, pH 7.7) wasprepared with RNase-free water to a final volume of 2.24 ml. Thereaction mixture was incubated at 37° C. for between 20-120 minutes.Upon completion, the mixture was treated with DNase I for an additional15 minutes and quenched accordingly.

The purified mRNA product from the aforementioned IVT step was denaturedat 65° C. for 10 minutes. Separately, portions of GTP (20 mM),S-adenosyl methionine, RNAse inhibitor, 2′-O-Methyltransferase andguanylyl transferase are mixed together with reaction buffer (10×, 500mM Tris-HCl (pH 8.0), 60 mM KCl, 12.5 mM MgCl₂) to a final concentrationof 8.3 ml. Upon denaturation, the mRNA was cooled on ice and then addedto the reaction mixture. The combined solution was incubated at 37° C.for 20-90 minutes. Upon completion, aliquots of ATP (20 mM), PolyAPolymerase and tailing reaction buffer (10×, 500 mM Tris-HCl (pH 8.0),2.5M NaCl, 100 mM MgCl₂) were added, and the total reaction mixture wasfurther incubated at 37° C. for about 20-45 minutes. Upon completion,the final reaction mixture is quenched and purified accordingly.

Example 2: Chromatographic Quantification of Capping Efficiency

This example demonstrates chromatographic quantification of capping aswell as methylation. Specifically, a twenty base DNA oligonucleotide isdesigned and synthesized to be complementary to the 5′ UTR of an invitro synthesized capped mRNA, specifically binding within one or twobases from the 5′ cap, and preferably binding adjacent to thepenultimate the mRNA (i.e., at the third nucleotide from the 5′ end, thelast two nucleotides of which are cap nucleotides. Binding of the DNAoligonucleotide to the 5′ UTR adjacent to the cap establishes awell-defined region of DNA:RNA hybrid that is susceptible to RNAseH-mediated cleavage. In some embodiments, the DNA oligonucleotide isflanked on either end by 1-15 RNA nucleotides. In some embodiments, theDNA oligonucleotide is flanked on the 3′ end by 1-15 RNA nucleotides.

RNAse H is added to a sample RNA solution comprising the hybridized invitro synthesized mRNA. The RNAase H cleaves the in vitro synthesizedcapped mRNA in the region of the DNA:RNA hybrid, thereby producing acapped fragment. In certain embodiments, this cleavage is accomplishedusing an S1 Nuclease or other nuclease to create a blunt-end fragmentmarked at the 5′ end by the cap analytes of choice. The fragment ispreferably 2-10 nucleotides in length, including the cap nucleotides.Overall, this process provides a smaller molecule with increasedresolution of both cap presence and cap modification.

After cleavage, the RNA solution is loaded onto a suitablechromatographic column for quantitative analysis of capped versusuncapped mRNA. In an exemplary embodiment, the column is ananion-exchange HPLC column, and the fragments are purified by adaptationof methods known to those of skill in the art (see, e.g., Wincott, F. etal, “Synthesis, deprotection, analysis and purification of RNA andribozymes), NUCLEIC ACIDS RES 23: 2677-2684 (1995); Anderson, A. C., etal., “HPLC purification of RNA for crystallography and NMR”, RNA, 2:110-117, (1996)). The amount of capped fragments in the sample isdetermined by integrating chromatographic peaks corresponding to thecapped and uncapped species. Data may be provided in the form of peakarea or ratio of capped to uncapped peak area.

Concurrently, using the same method for analysis, the percentmethylation of the guanine cap at the N-7 position is assessed. Evenfurther, when synthesizing a “Cap 1” structure motif, one could separatespecies which are methylated at the 2′-O position of the ribose ring ofthe penultimate base (FIG. 2). Measurement of methylated nucleotides byHPLC may be achieved by a variety of methods, including methodsincorporating electrochemical detection; see, e.g., Park and Ames (PROC.NATL. ACAD. SCI. USA, 85: 7467-7470 (1988)), incorporated by referenceherein. This methodology provides a powerful combination of quantitativecap assessment as well as quantitative methylation assessment.

Example 3: Evaluation of mRNA 5′ Capping on In Vivo Protein Production

In this example, we evaluated the impact of mRNA 5′ capping on in vivoprotein production and its potential impact on the efficacy of mRNAbased therapy. Specifically, we evaluated the impact of 5′ capping onthe in vivo production of alpha-galactosidase A (alpha-Gal A), which isdeficient in Fabry disease. Fabry disease is an X-linked inheritedlysosomal storage disease characterized by severe renal impairment,angiokeratomas, and cardiovascular abnormalities, including ventricularenlargement and mitral valve insufficiency. Fabry disease also affectsthe peripheral nervous system, causing episodes of agonizing, burningpain in the extremities. Fabry disease is caused by a deficiency in theenzyme alpha-galactosidase A (alpha-Gal A). alpha-Gal A is the lysosomalglycohydrolase that cleaves the terminal alpha-galactosyl moieties ofvarious glycoconjugates. Fabry disease results in a blockage of thecatabolism of the neutral glycosphingolipid, ceramide trihexoside (CTH),and accumulation of this enzyme substrate within cells and in thebloodstream.

The cDNA and gene encoding human alpha-Gal A, GLA, have been isolatedand sequenced. Human alpha-Gal A is expressed as a 429-amino acidpolypeptide, of which the N-terminal 31 amino acids are the signalpeptide. The human enzyme has been expressed in Chinese Hamster Ovary(CHO) cells (Desnick et al., U.S. Pat. No. 5,356,804; Ioannou et al., J.CELL BIOL., 119: 1137 (1992)); and insect cells (Calhoun et al., WO90/11353).

Individuals suffering from Fabry disease may be treated by enzymereplacement therapy with human alpha-Gal A (see, e.g., U.S. Pat. No.6,458,574, incorporated by reference herein). Additional approaches thatmodulate or supplement the expression of alpha-Gal A deficiency, andthus ameliorate the underlying deficiency, would be useful in thedevelopment of appropriate therapies for associated disorders. Suchapproaches include methods of intracellular delivery of nucleic acids(e.g., GLA mRNA) that are capable of correcting existing genetic defectsand/or providing beneficial functions to one or more target cells.Following successful delivery to target tissues and cells, thecompositions and nucleic acids transfect the target cell, and thenucleic acids (e.g., GLA mRNA) can be translated into the gene productof interest (e.g., alpha-GAL A) or can otherwise modulate/regulate thepresence or expression of the gene product of interest. Such methodshave been described previously; see, e.g. U.S. pre-grant publication2011/0244026, incorporated by reference herein.

In this example, we evaluated the impact of the 5′ capping on the invivo protein production. Human GLA mRNA was synthesized by in vitrotranscription from a plasmid DNA template encoding the gene, which wasfollowed by the addition of a 5′ cap structure, either Cap0 or Cap1(Fechter, P. et al., J. GEN. VIROLOGY, 86: 1239-1249 (2005)). A 3′poly(A) tail of approximately 200 nucleotides in length as determined bygel electrophoresis was also added. The 5′ and 3′ untranslated regionspresent in the GLA mRNA are represented as X and Y in SEQ ID NO: 7, asindicated below:

Alpha-galactosidase (GLA) mRNA (SEQ ID NO: 7):

XAUGCAGCUGAGGAACCCAGAACUACAUCUGGGCUGCGCGCUUGCGCUUCGCUUCCUGGCCCUCGUUUCCUGGGACAUCCCUGGGGCUAGAGCACUGGACAAUGGAUUGGCAAGGACGCCUACCAUGGGCUGGCUGCACUGGGAGCGCUUCAUGUGCAACCUUGACUGCCAGGAAGAGCCAGAUUCCUGCAUCAGUGAGAAGCUCUUCAUGGAGAUGGCAGAGCUCAUGGUCUCAGAAGGCUGGAAGGAUGCAGGUUAUGAGUACCUCUGCAUUGAUGACUGUUGGAUGGCUCCCCAAAGAGAUUCAGAAGGCAGACUUCAGGCAGACCCUCAGCGCUUUCCUCAUGGGAUUCGCCAGCUAGCUAAUUAUGUUCACAGCAAAGGACUGAAGCUAGGGAUUUAUGCAGAUGUUGGAAAUAAAACCUGCGCAGGCUUCCCUGGGAGUUUUGGAUACUACGACAUUGAUGCCCAGACCUUUGCUGACUGGGGAGUAGAUCUGCUAAAAUUUGAUGGUUGUUACUGUGACAGUUUGGAAAAUUUGGCAGAUGGUUAUAAGCACAUGUCCUUGGCCCUGAAUAGGACUGGCAGAAGCAUUGUGUACUCCUGUGAGUGGCCUCUUUAUAUGUGGCCCUUUCAAAAGCCCAAUUAUACAGAAAUCCGACAGUACUGCAAUCACUGGCGAAAUUUUGCUGACAUUGAUGAUUCCUGGAAAAGUAUAAAGAGUAUCUUGGACUGGACAUCUUUUAACCAGGAGAGAAUUGUUGAUGUUGCUGGACCAGGGGGUUGGAAUGACCCAGAUAUGUUAGUGAUUGGCAACUUUGGCCUCAGCUGGAAUCAGCAAGUAACUCAGAUGGCCCUCUGGGCUAUCAUGGCUGCUCCUUUAUUCAUGUCUAAUGACCUCCGACACAUCAGCCCUCAAGCCAAAGCUCUCCUUCAGGAUAAGGACGUAAUUGCCAUCAAUCAGGACCCCUUGGGCAAGCAAGGGUACCAGCUUAGACAGGGAGACAACUUUGAAGUGUGGGAACGACCUCUCUCAGGCUUAGCCUGGGCUGUAGCUAUGAUAAACCGGCAGGAGAUUGGUGGACCUCGCUCUUAUACCAUCGCAGUUGCUUCCCUGGGUAAAGGAGUGGCCUGUAAUCCUGCCUGCUUCAUCACACAGCUCCUCCCUGUGAAAAGGAAGCUAGGGUUCUAUGAAUGGACUUCAAGGUUAAGAAGUCACAUAAAUCCCACAGGCACUGUUUUGCUUCAGCUAGAAAAUACAAUGCAGAUGUCAUUAAAAGACUUACUUUAAY (SEQ ID NO: 4) X =GGGAUCCUACC  (SEQ ID NO: 6) Y = UUUGAAUU 

A codon-optimized alpha-galactosidase with PolyA insert (CO-GLA-PolyA)is also utilized in some embodiments (SEQ ID NO: 8):

X ₁AUGCAGCUGAGGAACCCAGAGCUCCAUCUCGGAUGUGCACUGGCACUUAGAUUUCUCGCGCUUGUGUCGUGGGACAUCCCCGGAGCCAGGGCGCUGGAUAAUGGGCUCGCCCGGACUCCCACAAUGGGUUGGCUGCACUGGGAGCGCUUUAUGUGCAAUCUGGACUGCCAGGAAGAGCCCGAUAGCUGUAUUUCGGAGAAGCUCUUCAUGGAAAUGGCGGAGUUGAUGGUGUCCGAAGGGUGGAAGGAUGCGGGAUAUGAGUAUCUGUGUAUCGAUGACUGCUGGAUGGCACCGCAGCGAGAUUCGGAGGGGCGAUUGCAGGCCGACCCUCAGCGCUUCCCUCAUGGAAUUCGGCAGCUGGCCAACUACGUACACUCAAAAGGACUUAAGUUGGGGAUCUACGCGGACGUCGGUAAUAAGACAUGCGCUGGGUUCCCGGGGAGCUUCGGAUACUAUGAUAUUGAUGCCCAGACCUUCGCGGACUGGGGAGUGGACUUGCUUAAGUUUGAUGGUUGUUACUGUGACUCAUUGGAAAACUUGGCGGAUGGGUAUAAACAUAUGUCCUUGGCCUUGAAUCGGACAGGGCGGUCGAUCGUCUACAGCUGCGAAUGGCCUUUGUAUAUGUGGCCGUUCCAGAAACCCAACUACACCGAAAUUCGCCAGUAUUGCAAUCACUGGAGAAACUUCGCCGAUAUCGACGAUUCGUGGAAAUCAAUCAAGUCCAUCCUCGACUGGACGUCCUUCAACCAAGAGAGAAUCGUAGAUGUGGCCGGACCGGGAGGAUGGAACGACCCUGAUAUGCUUGUAAUUGGCAACUUUGGACUCUCGUGGAACCAGCAAGUAACGCAAAUGGCACUCUGGGCUAUCAUGGCUGCGCCCCUGUUCAUGUCAAACGACCUCAGGCACAUCUCGCCGCAGGCGAAAGCCUUGCUUCAAGAUAAGGACGUCAUCGCGAUUAAUCAGGACCCGCUGGGGAAGCAGGGCUAUCAGCUUAGACAGGGCGACAAUUUUGAGGUCUGGGAGCGACCCCUGAGCGGACUCGCAUGGGCGGUGGCAAUGAUCAAUAGGCAGGAAAUUGGUGGGCCGAGGUCGUACACUAUCGCCGUCGCGUCGUUGGGAAAGGGUGUGGCGUGUAAUCCAGCGUGCUUUAUCACCCAACUGCUGCCCGUCAAGCGCAAACUGGGUUUUUACGAAUGGACGAGCAGACUUCGCUCACACAUUAACCCAACGGGUACGGUGUUGCUCCAGCUCGAGAAUACAAUGCAAAUGUCACUUAAAGAUUUGCUCUGACGGGUGGCAUCCCUGUGACCCCUCCCCAGUGCCUCUCCUGGCCCUGGAAGUUGCCACUCCAGUGCCCACCAGCCUUGUCCUAAUAAAAUUAAGUUGCAUCAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA

The GLA mRNA was stored in water at a final concentration of 1 mg/mL at−80° C. until the time of use. All mRNA concentrations were determinedvia absorption (γmax 260 nm).

Suitable formulations for in vivo delivery of GLA Cap0 mRNA, GLA Cap1mRNA, GLA mRNA and other controls include a multi-component lipidmixture of varying ratios employing one or more cationic lipids, helperlipids and PEGylated lipids. Cationic lipids can include (but notexclusively) DOTAP (1,2-dioleyl-3-trimethylammonium propane), DODAP(1,2-dioleyl-3-dimethylammonium propane), DOTMA(1,2-di-O-octadecenyl-3-trimethylammonium propane), DLinDMA (Heyes, J.;Palmer, L.; Bremner, K.; MacLachlan, I. “Cationic lipid saturationinfluences intracellular delivery of encapsulated nucleic acids”, J.CONTR. REL. 107: 276-287 (2005)), DLin-KC2-DMA (Semple, S. C. et al.“Rational Design of Cationic Lipids for siRNA Delivery”, NATUREBIOTECH., 28: 172-176 (2010)), C12-200 (Love, K. T. et al. “Lipid-likematerials for low-dose in vivo gene silencing”, PROC NATL ACAD SCI.,USA, 107: 1864-1869 (2010)), HGT4003, ICE, dialkylamino-based,imidazole-based, guanidinium-based, etc. Helper lipids can include (butnot exclusively) DSPC (1,2-distearoyl-sn-glycero-3-phosphocholine), DPPC(1,2-dipalmitoyl-sn-glycero-3-phosphocholine), DOPE(1,2-dioleyl-sn-glycero-3-phosphoethanolamine), DPPE(1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine), DMPE(1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine), DOPG(2-dioleoyl-sn-glycero-3-phospho-(1′-rac-glycerol)), cholesterol, etc.The PEGylated lipids can include (but not exclusively) a poly(ethylene)glycol chain of up to 5 kDa in length covalently attached to a lipidwith alkyl chain(s) of C6-C20 length. Lipid encapsulation of mRNA wascalculated by performing the Ribogreen assay with and without thepresence of 0.1% Triton-X 100. Particle sizes (dynamic light scattering(DLS)) and zeta potentials were determined using a Malvern Zetasizerinstrument in 1×PBS and 1 mM KCl solutions, respectively.

Aliquots of 50 mg/mL ethanolic solutions of C12-200, DOPE, Chol andDMG-PEG2K were mixed and diluted with ethanol to 3 mL final volume.Separately, an aqueous buffered solution (10 mM citrate/150 mM NaCl, pH4.5) of CO-GLA mRNA (Cap 0 or Cap 1) was prepared from a 1 mg/mL stock.The lipid solution was injected rapidly into the aqueous mRNA solutionand shaken to yield a final suspension in 20% ethanol. The resultingnanoparticle suspension was filtered, diafiltrated with 1×PBS (pH 7.4),concentrated and stored at 2-8° C. Final concentration=0.72 mg/mL GLAmRNA (encapsulated). Zave=85.5 nm (Dv(50)=61.9 nm; Dv(90)=113 nm).

To determine whether the type of cap incorporated into GLA mRNAinfluenced protein production when the mRNA was encapsulated intoC12-200-based lipid, an experiment was conducted in which wild type(CD-1) mice were injected with capped GLA mRNA species and subsequentlymonitored for human GLA protein production. The capped mRNA speciesincluded Cap0 (unmethylated at the 2′-O position) and Cap1(2′-Omethylated)

The foregoing studies were performed using male CD-1 mice ofapproximately 6-8 weeks of age at the beginning of each experiment.Samples were introduced by a single bolus tail-vein injection of anequivalent total dose of 30 micrograms of encapsulated GLA, EPO, FIX orAIAT mRNA. Serum concentrations of GLA protein were determined at sixhours. All animals were euthanized by CO₂ asphyxiation 6 hours post-doseadministration (±5%) followed by thoracotomy and terminal cardiac bloodcollection. Whole blood (maximal obtainable volume) was collected viacardiac puncture on euthanized animals into serum separator tubes,allowed to clot at room temperature for at least 30 minutes, centrifugedat 22° C.±5° C. at 9300 g for 10 minutes, and the serum was extracted.For interim blood collection at six hours, approximately 40-50 μL ofwhole blood was be collected via facial vein puncture or tail snip.Samples collected from non-treatment animals were used as a baseline GLAlevels for comparison to study animals. The liver and spleen of eachmouse was harvested, apportioned into three parts and stored in either10% neutral buffered formalin or snap-frozen and stored at 80° C.

Human GLA protein production was measured by enzyme-linked immunosorbentassay (“ELISA”). Standard ELISA procedures were followed employing sheepanti-Replagal G-188 IgG as the capture antibody with rabbitanti-Replagal IgG as the secondary (detection) antibody. Horseradishperoxidase (HRP)-conjugated goat anti-rabbit IgG was used for activationof the 3,3′,5,5′-tetramethylbenzidine (TMB) substrate solution. Thereaction was quenched using 2N H₂SO₄ after 20 minutes. Detection wasmonitored via absorption (450 nm) on a Molecular Device Flex Stationinstrument. Untreated mouse serum and human Replagal® protein were usedas negative and positive controls, respectively.

As illustrated in FIG. 3, following the intravenous injection of cappedspecies of CO-GLA mRNA loaded in the C12-200-based lipid nanoparticles,a substantial level of human GLA protein could be detected in mouseserum within 6 hours. Notably, there was a dramatic and statisticallysignificant increase in protein production when employing mRNA with aCap1 structure versus that of a Cap0 structure. These resultsdemonstrated the importance of having the ability to characterize andquantify the capping and methylation efficiencies of the mRNA synthesisprocess.

EQUIVALENTS AND SCOPE

Those skilled in the art will recognize, or be able to ascertain usingno more than routine experimentation, many equivalents to the specificembodiments of the invention described herein. The scope of the presentinvention is not intended to be limited to the above Description, butrather is as set forth in the appended claims.

In the claims articles such as “a”, “an” and “the” may mean one or morethan one unless indicated to the contrary or otherwise evident from thecontext. Thus, for example, reference to “an antibody” includes aplurality of such antibodies, and reference to “the cell” includesreference to one or more cells known to those skilled in the art, and soforth. Claims or descriptions that include “or” between one or moremembers of a group are considered satisfied if one, more than one, orall of the group members are present in, employed in, or otherwiserelevant to a given product or process unless indicated to the contraryor otherwise evident from the context. The invention includesembodiments in which exactly one member of the group is present in,employed in, or otherwise relevant to a given product or process. Theinvention includes embodiments in which more than one, or all of thegroup members are presenting, employed in, or otherwise relevant to agiven product or process. Furthermore, it is to be understood that theinvention encompasses all variations, combinations, and permutations inwhich one or more limitation, elements, clauses, descriptive terms,etc., from one or more of the listed claims is introduced into anotherclaim. For example, any claim that is dependent on another claim can bemodified to include one or more limitations found in any other claimthat is dependent on the same base claim. Furthermore, where the claimsrecite a composition, it is to be understood that methods of using thecomposition for anyone of the purposes disclosed herein are included,and methods of making the composition according to any of the methods ofmaking disclosed herein or other methods known in the art are included,unless otherwise indicated or unless it would be evident to one ofordinary skill in the art that a contradiction or inconsistency wouldarise.

Where elements are presented as lists, e.g., in Markush group format, itis to be understood that each subgroup of the elements is alsodisclosed, and any element(s) can be removed from the group. It shouldbe understood that, in general, where the invention, or aspects of theinvention, is/are referred to as comprising particular elements,features, etc., certain embodiments of the invention or aspects of theinvention consist, or consist essentially of, such elements, features,etc. For purposes of simplicity those embodiments have not beenspecifically set forth in haec verba herein. It is noted that the term“comprising” is intended to be open and permits the inclusion ofadditional elements or steps.

Where ranges are given, endpoints are included. Furthermore, it is to beunderstood that unless otherwise indicated or otherwise evident from thecontext and understand of one of ordinary skill in the art, values thatare expressed as ranges can assume any specific value or sub-rangewithin the state ranges in different embodiments of the invention, tothe tenth of the unit of the lower limit of the range, unless thecontext clearly dictates otherwise.

In addition, it is to be understood that any particular embodiment ofthe present invention that falls within the prior art may be explicitlyexcluded from any one or more of the claims. Since such embodiments aredeemed to be known to one of ordinary skill in the art, they may beexcluded even if the exclusion is not set forth explicitly herein. Anyparticular embodiment of the compositions of the invention can beexcluded from any one or more claims, for any reason, whether or notrelated to the existence of prior art.

The publications discussed above and throughout the text are providedsolely for their disclosure prior to the filing date of the presentapplication. Nothing herein is to be construed as an admission that theinventors are not entitled to antedate such disclosure by virtue ofprior disclosure.

Other Embodiments

Those of ordinary skill in the art will readily appreciate that theforegoing represents merely certain preferred embodiments of theinvention. Various changes and modifications to the procedures andcompositions described above can be made without departing from thespirit or scope of the present invention, as set forth in the followingclaims.

We claim:
 1. A method of manufacturing mRNA having a quantifiedpercentage of capped mRNA comprising the steps of: (1) manufacturingmRNA by in vitro synthesis to generate in vitro synthesized mRNAcomprising capped mRNA and uncapped mRNA; (2) obtaining a sample of thein vitro synthesized mRNA; (3) annealing the mRNA sample with a DNAoligonucleotide complementary to a sequence in the 5′ untranslatedregion of the in vitro synthesized mRNA, wherein the first base of the3′ end of the DNA oligonucleotide binds within 5 bases from the cap oruncapped penultimate base of mRNA under conditions that permit the DNAoligonucleotide to anneal to the sequence and form a DNA/RNA hybrid; (4)introducing to the sample one or more nucleases that selectivelydegrades the DNA/RNA hybrid, resulting in capped and uncapped fragmentscomprising no more than 5 bases of the mRNA; (5) separating the cappedand uncapped fragments in the sample by chromatography and measuring therelative peak areas of the capped and uncapped fragments; and (6)determining the relative amount of the capped and uncapped fragments inthe sample by determining the relative peak areas of the capped anduncapped fragments, thereby quantifying the percentage of capped mRNA.2. The method of claim 1, further comprising a step of releasing themanufactured mRNA lot.
 3. The method of claim 1, wherein the capped mRNAcomprises a cap that has a structure of formula I:

wherein, B is a nucleobase; R₁ is selected from a halogen, OH, and OCH₃;R₂ is selected from H, OH, and OCH₃; R₃ is CH₃, CH₂CH₃, CH₂CH₂CH₃ orvoid; R₄ is NH₂; R₅ is selected from OH, OCH₃ and a halogen; n is 1, 2,or 3; and M is a nucleotide of the mRNA.
 4. The method of claim 3,wherein the nucleobase is guanine.
 5. The method of claim 1, wherein thecapped mRNA comprises a cap that is a m⁷G cap with a structure offormula II or an unmethylated cap with a structure of formula III:

wherein, R₂ is H or CH₃; R₄ is NH₂; R₅ is OH or OCH₃; R₆ is H or CH₃,and M is a nucleotide of the mRNA; or

wherein M is a nucleotide of the mRNA.
 6. The method of claim 1, whereinstep (5) further separates methylated and unmethylated capped RNA. 7.The method of claim 6, wherein the methylated cap comprises methylationat R₃ and/or R₅ position.
 8. The method of claim 6, wherein step (6)further comprises a step of quantitatively determining methylationpercentage of the capped RNA.
 9. The method of claim 1, wherein the DNAoligonucleotide is 10-80 nucleotides in length.
 10. The method of claim1, wherein the DNA oligonucleotide is linked to one or more RNAnucleotides.
 11. The method of claim 1, wherein the one or morenucleases comprise RNase H.
 12. The method of claim 1, wherein the oneor more nucleases comprise a nuclease that generates blunt-ended cappedand uncapped fragments.
 13. The method of claim 12, wherein the nucleaseis nuclease S1, RNAse H, or another 5′ exonuclease.
 14. The method ofclaim 1, wherein the capped and uncapped fragments comprise no more than2 bases of the mRNA.
 15. The method of claim 1, wherein thechromatography in step (5) is achieved with a chromatographic columnselected from the group consisting of an anion-exchange HPLC column, acation-exchange HPLC column, a reverse phase HPLC column, a hydrophobicinteraction column, an ultra-performance liquid chromatography column,or a size exclusion column.